Efficient and modular CRISPR-Cas9 vector system for Physcomitrella patens.

Area of Science:

• Plant biotechnology
• Genome editing
• Molecular biology

Background:

• CRISPR-Cas9 enables precise genome editing via RNA-guided nucleases.
• Homologous recombination is a traditional gene targeting method in *Physcomitrella patens*.
• CRISPR-Cas9 demonstrates higher efficiency than homologous recombination in *P. patens*.

Purpose of the Study:

• To create a modular CRISPR-Cas9 vector system for *P. patens* genome editing.
• To enable simultaneous editing of multiple loci and precise DNA sequence integration.
• To facilitate efficient gene targeting and allele generation.

Main Methods:

• Development of a flexible and modular CRISPR-Cas9 vector system.
• Transient transformation for genome editing.
• Generation of DNA donor plasmids for homology-directed repair.
• Use of a "stop codon cassette" for null allele generation.

Main Results:

• The vector system allows simultaneous editing of up to 12 loci without gene synthesis.
• Multiple null alleles were generated at four distant loci.
• Targeting multiple sites within a locus can create larger deletions, depending on protospacers.
• Cas9 cleavage of the DNA donor plasmid reduces homology-directed repair efficiency if the protospacer is present.
• A homology plasmid with a "stop codon cassette" facilitates null allele generation and genotyping.

Conclusions:

• The developed CRISPR-Cas9 vector system significantly enhances genome editing efficiency in *P. patens*.
• The system supports multiplex genome editing and precise DNA integration.
• This tool advances functional genomic studies in *P. patens* and other plant species.